The focus of our lab has been to use our knowledge of tolerance mechanisms to develop novel approaches for the prevention or reversal of undesirable immune responses, including inhibitor formation in hemophilia. Up to 25-30% of hemophilia patients produce antibodies (inhibitors) to therapeutic clotting factor VIII (FVIII), presumably because tolerance has not been adequately induced. Therefore, the induction of tolerance to FVIII is an important goal. We have utilized immunoglobulin (Ig) fusion proteins delivered via retroviral vectors in B-cells for the induction of tolerance. The multiple epitopes so expressed via B-cell MHC class II antigen presentation have been shown to lead to immunologic tolerance at both T-cell and B-cell levels. Data in several experimental autoimmune models (uveitis, EAE for multiple sclerosis and diabetes) are promising in that significant clinical efficacy has been achieved, even in immunized recipients. While effects on antibody-mediated responses have been obtained with model antigens, extension to modulation of undesirable T-cell dependent antibody responses in a mouse model for hemophilia has not been tested. The novelty of this system is that the target antigen, FVIII, is well defined, as are the major domains recognized by inhibitors. Herein we propose to apply this technology for the prevention and/or elimination of FVIII inhibitors in murine hemophilia. Our initial goal is to induce tolerance to major domains of FVIII, to evaluate the requirement for the Ig scaffold, and to analyze the cellular basis of tolerance, including the role of CD25 + regulatory T cells. Specifically, we will engineer multiple domains of FVIII (A2, C2) into retroviral fusion protein constructs delivered singly or in concert via B-cells for effective tolerance induction, as measured by both T-cell and B-cell responsiveness. Importantly, we will determine whether this approach will lead to the elimination of responses not only to those domains, but also to the entire therapeutically delivered molecule. Later, we will apply this approach to reverse ongoing immune responses to FVIII in primed FVIII knockout (-/-) mice, and determine whether anti-CD40L treatment enhances the tolerogenicity in this model. The fate, activation state and responsiveness of domain-specific primed T cells will be followed to assess the mechanisms of tolerance. Using modified lentiviral vectors, we will test whether expression of B-domain less FVIII may be necessary to achieve effective tolerance, and if the IgG carrier enhances this process. These studies are an important step toward future pre-clinical testing in non-human primates.